Apparatus and method for early diagnosis of cell death

ABSTRACT

An apparatus for measuring through optical means temporally resolved, optical properties, and/or phenotypes, linked to cellular homeostasis. Those temporal measurements enable the detection of cell regulation through various channels linked to homeostasis, in order to assess cell viability or early cell death through rapid diagnostic.

FIELD OF INVENTION

The present invention relates to methods and apparatuses for detectingthe state (alive or death) of a cell or a group of cells.

BACKGROUND OF THE INVENTION

The detection of cell death is a highly relevant biomarker for manybiological processes related to various application fields including theseverity/progression of diseases, the efficacy of various therapies andthe drug safety evaluation. Cell death can proceed by several distinctpathways, including mainly apoptosis, necrosis and autophagy, which arecharacterized by a distinct set of temporal, morphological, biochemical,and genetic characteristics. For a detailed description of the variousmechanisms involved in the different cell death pathways, see forexample Duprez, L. et al. “Major cell death pathways at a glance”,Microbes Infect. 11(13), 1050-1062 (2009).

Although, in a whole organism, certain types of cell death, includingapoptosis, result in the controlled breakdown of the cell avoiding anyintracellular release, in vitro, the different cell death pathwaysproceed to an end-stage called secondary necrosis, which shares manyfeatures with primary necrosis pathway, in particular the loss of cellmembrane integrity and the subsequent release of the cellular contentinto the surrounding extracellular space. Consequently, in vitro, assaysusually differentiate between viable and non viable cells by assessingmembrane integrity thanks to inclusion and/or exclusion dyes (trypanblue or propidium iodide, for example) or the detection of specificintracellular compounds in the surrounding medium (lactatedeshydrogenase release (LDH)). However, depending on the stimulus havinginduced cell death, such cell viability essays assess a late stage ofthe cell death processes with extrinsic contrast agents and usuallyrequire several steps (washing, harvesting, solubilization etc.) whichtake several hours for completion.

On the other hand, as mentioned before, specific morphological andbiochemical features (often called “phenotypes”) accompany or are linkedto cell death processes and are often used to define and recognize thedifferent cell death pathways. For example, the loss of cell volume orcell shrinkage that occurs during apoptosis is a key morphologicalcharacteristic separating this physiological cell death process from anaccidental one as necrosis, characterized by an initial cell swelling.Originally, the volume regulation is driven by homeostasis, which is theconcept of the cell regulating within its environment. On a generalpoint of view, a combination of various parameters and phenotypes linkedto the homeostasis, such as protein concentration, ion concentration,water content, etc., can provide useful indicators about the cellviability.

It was indeed shown that loss of the cell normal regulatory capabilityis considered as a trigger for cell death. For example, cell volumederegulation was shown to be a relevant indicator for cell deathtriggering, and intracellular ionic concentration deregulation is seenas a primary indicator of cell biological processes dysfunction, asshown for example in Bortner, C. and Cidlowski, J., “The role ofapoptotic volume decrease and ionic homeostasis in the activation andrepression of apoptosis,” Pflug. Arch. Eur. J. Physiol. 448(3), 313-318(2004). However, minor variations of those parameters occur inaccordance with the normal cell activity and cells have inheritedregulatory mechanisms to compensate for these minor variations in orderto maintain in particular appropriate balance of ions across their cellmembrane. One can thus identify, for a given regulatory indicator, avariation range which lies in normal life cycle of the cell, and otherranges indicating a lack of regulation capacity of the cell, which canlead to cell death mechanisms triggering.

GENERAL DESCRIPTION OF THE INVENTION

The present invention makes use of the direct relationship that linksoptical properties to intracellular concentrations (e.g. water content,protein concentration, ions concentrations) and to cellular morphology(e.g. cell volume, cell surface, cell thickness). As a result of theserelationships, optical measurements can be used as a direct mean tomonitor the regulatory behavior of cells. In other words, the presentinvention uses temporally resolved measurement of optical properties, inorder to define phenotypes enabling to monitor the ability of cells torestore their homeostatic equilibrium after a perturbation of theirenvironment. Such optical monitoring enables non-invasive, possiblyearly diagnosis of cell death.

The invention more precisely concerns a method and an apparatus asdefined in the claims.

The aim of the proposed invention is to provide a method and anapparatus measuring cell regulatory parameters and phenotypic evolutionthrough time, in order to enable a rapid or time-efficient diagnostic ofcell viability. Measuring a given regulatory property, such as forexample volume, intracellular ionic concentration, intracellular watercontent, proteins concentrations, morphology, or more generally acombination of phenotypes in a time-resolved manner enables to determinethe cell viability with a high sensitivity and temporal resolution, thatcan usually lead to a rapid and early diagnostic.

The invention describes an apparatus performing the measurement throughoptical means, so that the regulatory property, or phenotype, or acombination of them, of interest is deduced from the measurement of anelectromagnetic wave that interacted with the bio-sample, thus providinga non-invasive measurement. The apparatus comprises a processing unit,which deduces the optical parameter of interest from the measurement ofthis said electromagnetic wave. The apparatus comprises also an analysisunit providing real-time processing of the measurement, in order todeduce the temporal change of the measured regulatory parameters. Fromthis temporal measurement, a post-processing unit is capable of deducinga diagnostic about cell viability, by processing the time-resolvedmeasurement in order to provide a decision, that can be eitherimplemented in quasi real-time to get fast and early diagnostic, or as apure data processing step to be performed anytime a-posteriori.

As the measurement is performed globally on the whole sample throughoptical means, the detection can be employed on single cellsindividually, on cell cultures as a whole, as well as biologicaltissues.

As mentioned previously the invention concerns an apparatus and a methodfor fast and early diagnosis of cell death by time-resolved measurementof an optical parameter, said time-resolved measurement of phenotypesbeing an indicator of the ability of cells to restore, by spontaneousregulation, their homeostatic equilibrium after a perturbation of theirenvironment. The speed advantage of the approach does not precludelong-duration experiments or a-posteriori data processing depending ofthe chemical agent, cell type or experimental requirements. If themeasurement of the optical parameter indicates a reversible behaviorover time, cells are considered as alive. On the contrary, if themeasurement of the optical parameter indicates a non-reversible behaviorover time, cells are considered as dead. The measured optical parametercan be for example the optical phase, or the optical path length, whichare of particular relevance to monitor cellular regulation behaviors,since these parameters are directly related to the refractive index ofthe cell, an optical property which is highly sensitive to intracellularconcentrations of proteins or ions, or water content. In addition,optical phase and optical path length are also highly sensitive tomorphological properties (cell thickness, volume, projected area, etc. .. . ) and intra-cellular refractive index (linked to e.g. dry-mass,water content, etc. . . . ). Within the frame of the present inventionall these parameters can define phenotypes, that can be either linked toa physical metric (e.g. volume, projected area, intracellular refractiveindex, . . . ) or to a cell culture behavior over time (e.g. mytosisrate, motility, culture grow patterns, etc. . . . ), to be evaluated andtracked over time, either individually or in combination, to finally geta time-efficient viability diagnostic based on their reversibility.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the scheme of an apparatus for measuring, in atime-resolved way, an optical property employed as an indicator ofhomeostasis on a bio-sample for fast cell death detection.

FIG. 2 presents schematically some examples of possible responses from ameasurement of an optical property through time on a bio-sample,according to the present invention. (a) The signal drops, but regulationoccurs. (b) The signal drops and cannot be compensated throughregulation. (c) The signal increases, but regulation occurs. (d) Thesignal increases and cannot be compensated through regulation.

FIG. 3 presents schematically a transmission digital holographicmicroscope that can be used to measure, in a time resolved way, anoptical property for early diagnosis of cell death according to thepresent invention. BS: beamsplitter, M: Mirror C: Condenser lens, MO:Microscope objective, FL: Field lens, IM: Image plane. A computeranalyses the signal detected at the CCD camera to retrieve the opticalparameter.

FIG. 4 presents an example of one embodiment of the present inventionwith a measurement of cell regulation measured through digitalholographic microscopy for cell death detection, in order to show thevalidity of the approach. (a) Curve of the optical phase measured as afunction of time, when a drug is employed to induce cell death. In thisparticular case, cell 4 shows clearly a volume regulation, indicatingthat this particular cell survived the drug exposure. This behavior hasbeen confirmed with an assessment dye (trypan blue) by (b) firstchecking cell morphology before the experiment, and then (c) severalhours after the experiment, showing that cell 4 is not stained by thedye, while the other dead cells are stained.

FIG. 5 shows examples of curves of time-resolved phase values measuredthrough digital holographic microscopy, where (a) cells survive the drugexposure, showed through the signal reversibility, or where (b) cells donot survive the drug exposure, as it is indicated by thenon-reversibility of the signal.

DETAILED DESCRIPTION OF THE INVENTION

The basic principle of the described invention relies on a time-resolvedmeasurement of one (or several) optical property(-ies), saidtime-resolved measurement of phenotypes being used as an indicator ofcell viability. More precisely and simply described, the presentinvention analyses the reversibility, or the non-reversibility, oftemporal variations of one, or a combination of, phenotype(s) based onthe measurement of an optical property of a biological sample, andprovides a diagnosis of cell death on the basis of this analysis, saiddiagnosis being based on the following considerations:

-   -   Reversible variations indicate that a regulation process        occurred to restore the homeostatic equilibrium of cells,        indicating that the cell can be considered as alive.    -   Non-reversible variations, whose amplitudes are higher than        expected for a standard physiological behavior, are the        indicator of cellular death.

Two general principles govern cell death diagnosis according to thepresent invention:

-   -   First a biological principle that links the cell death process        to cellular regulation processes: Simply explained, after a        perturbation of its environment, e.g. the exposition to a toxic        substance, a cell that succeeds to regulate itself to restore a        state of equilibrium with respect to its environment (further        called homeostatic equilibrium), is a cell that can be        considered as a healthy or living cell. On the contrary, a cell        that fails in restoring its homeostatic equilibrium can be        considered as dead, or at least, his future death can be        diagnosed. This first principle explains how an analysis of        reversibility/non-reversibility, can be used to diagnose cell        death.    -   Second a physical principle that links the optical properties of        a cell to cellular regulation processes: Indeed, among the        diverse forms of regulation processes that a cell can use to        adapt itself to a perturbation of its environment, several forms        of regulation affect for example the cellular morphology (e.g.        the cell volume, the projected cell area, etc. . . . ) and/or        the intracellular concentrations (e.g. proteins or ions        concentrations, water content, dry mass, nucleus/DNA        condensation, etc. . . . ). As a consequence, the optical        properties of a cell are modified by a regulation process, since        optical measurement methods are sensitive to both morphological        parameters and intracellular concentrations. For example,        changes of intracellular concentrations induce automatically        changes of the refractive index of the cell, which modifies the        speed of propagation of electromagnetic waves (optical waves in        particular) traveling through the cell. This second principle        explains how temporally-resolved measurement of an optical        property can define various phenotypes that can be used to        monitor a cell regulation process, thus enabling a diagnosis of        cell death, according to the first principle explained before.        These two principles will be described in more details in what        follows, and a concrete implementation of the present invention        will be described.

Cells regulate themselves within their environment through homeostasis,depending on the extracellular concentrations of biochemical compounds.For instance, osmosis will induce water movements through the cellmembrane, thus inducing changes in ionic concentrations, and volumevariations. The proposed apparatus is based on temporal measurement ofsuch parameters, or more generally any phenotype that can be retrievedthrough optical path length measurement, in order to measure theregulation capabilities of the cellular body. Small variations areconsidered to be physiologically relevant in a healthy process of thecell life cycle, as it preserves its environment through homeostasis. Onthe other hand, strong changes which cannot be regulated throughstandard channels lead to internal deregulation, and ultimately triggercell death mechanisms, during which a cascade of phenomena occurs. Themeasurement principle therefore relies on the detection of suchderegulations for an early diagnostic of cell viability. The mainadvantage of this approach is to provide a faster way of detection, asderegulations (biochemical, volume, morphological phenotypes, etc.) arethe primary causes for triggering the cell death mechanisms. Detectingsuch deregulations through their phenotypic signatures makes it thuspossible to detect the cause of cell death triggering instead ofconsequences of cell death, such as cell viability assessments based onextraneous dyes relying on cell permeability, which occur when aspecific type of cell death—necrosis—is at its late stages.

One can cite many different changes in the homeostasis of a cell whendeath pathways are triggered. We give here a description of some of themost typical phenotypes, although this description is not meant to beexhaustive. In the case of apoptosis, which corresponds to thecontrolled death aiming at recycling cell organelles without releasingintracellular compounds into the surrounding medium, one can cite cellshrinkage, increase of the intracellular ionic or proteinconcentrations, important and rapid morphological variations of themembrane surface, adenine triphosphate (ATP) depletion, etc. Then, thecase of necrosis, which corresponds to the uncontrolled pathologicaldeath, is characterized by cell swelling, loss of morphology controlleading to spherical shape, dilution of the intracellular content,increase of the cell membrane permeability, etc. Finally, autophagy is anormal process occurring as suppression of organelles inside the celland selection of intracellular compounds for preservation of thestability of the genome. In the context of cell death, it ischaracterized by an absence of fragmentation of the nucleus until latestage, strong lysosomic activity and no division of the cell body inseveral small compartments.

However, in usual cases, the different pathways leading ultimately tocell death are in competition, so that many different variations inregulation occur, and that one parameter only cannot be employed todiagnose a specific type of death. Nevertheless, all those events can berelated to changes in homeostasis, and can be employed in a global wayfor fast detection.

In its most simple implementation, the invention is based on anapparatus employing the elements described below, and shown in FIG. 1.An emitter (101) generates an electromagnetic wave, which passes throughthe sample (102). After having interacted with the sample, the wave ismeasured with a detector (103), which sends the measured signal to aprocessing unit (104). The processing unit converts the measured signalto one, or several, optically-resolved phenotype(s) of interest (105),such as cell volume or intracellular compound concentration, forinstance. This phenotype, or a combination of several phenotypes, is/arethen stored and processed during time by an analysis unit (106),providing the time-resolved monitoring of the phenotype(s) of interest(107), which can then be processed for analysis by a detection unit(108), which finally provides a diagnostic (109) through analysis of thetemporally resolved phenotype(s).

In its practical implementation, the apparatus should include ameasurement device to measure a given optical property corresponding toa specific phenotype, and a processing unit to derive and analyze themeasured data. Due to the various phenotypes which can be measured toestimate the regulation capability of cells, many different opticalmeans can be developed for this purpose. To estimate for instance thevolume of cells, interferometry can be employed through the measurementof the optical path length induced by cells, such as in white lightinterferometry, holography, or digital holography, or through z-resolvedmeasurements, such as with confocal microscopy, optical coherencemicroscopy, phase retrieval through z-stack of intensity images(transport-of-intensity), or other scanning or phase retrieval methods,provided that the scanning procedures still enables a temporalmonitoring. To estimate for instance ionic or protein content, one coulduse interferometry or refractometers, where the refractive index isrelated to the optical path length induced by the cell, or functionalimaging such as fluorescence microscopy, where specific fluorophoresenable the measurement of specific intracellular compounds. Theprocessing unit can be for instance a computer with dedicated imagepost-processing software working either on classical central processingunits (CPU) or on graphical processing units (GPU) for parallelcomputing, or a dedicated hardware (such as a PCI board) for directprocessing. Typically, either online computation of the temporal dataand diagnosis (simultaneous to measurement) or offline (aftermeasurement) could be performed depending on the specificimplementation.

Typical temporal signals that can be obtained through this apparatus areshown schematically in FIG. 2, for different responses of thebio-sample. In FIG. 2(a-b), the case where the optical propertydecreases under stimulation (chemical, electrical, etc.) is presented.In FIG. 2(a), the optical property measurement is recovering to a levelcomparable to before the stimulation, where the difference Δ can beconsidered as being within physiological variations, indicating that thecell regulated itself, and can thus be considered as viable. On theother side, in the case of FIG. 2(b), the decrease is not compensatedthrough regulation phenomena, so that no increase after the end of thestimulation is seen, and the difference of the optical property Δ cannotbe considered as being in physiological ranges, so that the cell is notviable. On the other hand, FIG. 2(c-d) presents a similar case, butwhere the optical property measured on the cell response is increasingthrough stimulation. In FIG. 2(c), the regulation is occurring andcompensating for the changes, showing again the cell viability, while inFIG. 2(d), the regulation is not compensating for the changes,demonstrating that the cell is non-viable.

Depending on the type of processing unit employed, the detection can beapplied to various types of bio-samples. Typically, the detection can beapplied at a single cell level, but different type of processing canlead to an interpretation of the phenotypes at a more global level, sothat direct detection on a cell culture or a biological tissue could beemployed in a perspective of statistical treatment, for instance.

Feasibility Demonstration

We present in this part of the document a validation of the detectionapproach described above. In this particular case, the implementation ofthe apparatus is an interferometric measurement device based on digitalholographic microscopy (DHM), as described for example by E. Cuche andC. Depeursinge in EP1119798—METHOD AND APPARATUS FOR SIMULTANEOUSAMPLITUDE AND QUANTITATIVE PHASE CONTRAST IMAGING BY NUMERICALRECONSTRUCTION OF DIGITAL HOLOGRAMS (2000), and by T. Colomb et al. inWO2006090320—Wave Front Sensing Method And Apparatus (2006). In EP1451646—APPARATUS AND METHOD FOR DIGITAL HOLOGRAPHIC IMAGING (2003), P.Marquet et al. present an application of the method in biology thatdescribes quantitative phase-contrast imaging in biology.

The particular implementation is a transmission DHM setup makingpossible to monitor cells in time through phase measurement. The sketchof the optical arrangement of the measurement setup is shown in FIG. 3,where the light emitted by a light source is split into two beams andrecombined to generate an interference producing a hologram on thecamera. This interference occurs between a reference wave that do notinteract with the sample, and an object wave collected by a microscopeobjective (MO) after interaction with the sample. Phase images arereconstructed by a numerical method for off-axis digital hologramreconstruction.

DHM is of particular interest for implementing an apparatus according tothe present invention because it enables a fast and easy access to thephase information of the object wave, which is of particular relevanceto probe cell regulation processes and to define a variety of phenotyperelated to morphology, intracellular content or cell membrane propertiesfor example. DHM also enables a fully automated access to the phaseinformation. But it is clear that the present invention is notrestricted to the use of a transmission DHM, as described as a pureexample in FIG. 3. It is also clear that optical components such aslenses, mirrors, wave-plates and prisms can be added on the generalsetup of FIG. 3.

In the different experiments described below, the optical property,employed for cell death detection is the mean phase value measured oncell bodies and monitored during time. The phase value is intrinsicallya relative measurement that has to be compared with a phase reference,usually taken as the phase shift induced by the perfusion medium.Physically, the phase value φ can thus be related to an optical pathlength (OPL) as

$\begin{matrix}{{{{\phi_{i}\left( {x,y} \right)} - {\phi_{m}\left( {x,y} \right)}} = {{{\Delta\phi}\left( {x,y} \right)} = {\frac{2\pi}{\lambda}\left( {n_{i} - n_{m}} \right){h\left( {x,y} \right)}}}},} & (1)\end{matrix}$

where the indices i and m correspond respectively to the intracellularcontent of the cell and to the perfusion medium, h is the height of thecell at position (x, y) and n is the mean refractive index (RI) alongthe optical axis. The phase is measured by calculating a spatialaveraging of the phase value on a constant surface contained in the cellbody, to which a reference phase value measured on a zone of the fieldof view containing no biological material has been subtracted.

The meaning of the phase measured with an interferometric method in thiscontext is dependent on the height of the cell and on its intracellularRI. Therefore, the phase is linked both to the cell volume phenotype(through cell height), and to the intracellular content of cellsphenotype (through RI), and thus indirectly to the osmotic regulation ofthe cell. The phase signal is therefore an indicator of cell volumeregulation through both parameters, and can be indirectly linked tohomeostasis through osmotic phenomena.

This particular study is performed on primary cultures of mouse corticalneurons, mounted on coverslips for optical measurement. Beforeexperiment, coverslips are mounted on a perfusion chamber used to applythe different solutions to the cells, which are immersed in aHEPES-buffered standard physiological perfusion medium. As an example ofapplication of cell death detection through volume regulation, wepresent in the following a detection of neuronal death induced throughglutamate-induced excitotoxic stimulation, ultimately leading to celldeath. The glutamate is a neurotransmitter activating neuronalreceptors, leading under strong exposure to high level of intracellularcalcium concentration, which can lead to death pathway triggering if notregulated properly.

In order to validate the measurement principle proposed in thisapplication, the cell viability is also tested with trypan blue 0.4%reagent, to enable comparative measurements. The dye relies on probingthe cell membrane integrity, which becomes permeable to the colorcompound at a certain level after cell death mechanisms started. Afterreagent wash-out, the cell nuclei of non-viable cells are consequentlystained in blue, as the dye fixes on DNA material. Excitotoxic effectswere studied during the experiments, by applying glutamate pulses atdifferent concentration to the cells. The excitation solutions wereprepared with concentrations ranging between 25 μM and 100 μM, dependingon the type of experiment. Perfusion changes were performed by washingthe chamber with a micropipette, making it possible to replace theperfusion medium through typically two washes of the medium.

In order to enable color measurement for cell viability assessment withthe reagent, a flip mirror is inserted after the MO, so that theintensity image could also be recorded in focus with a color camera.When performing trypan blue staining, the illumination is changed toemploy an incoherent halogen white light source to enable colormeasurement; this implies that during dye probing, no DHM measurement isperformed, yielding part of unmeasured phase signal during timemonitoring. Image merging between the two measurement techniques couldbe done through calibration of the system by imaging an object whoseshape is well-known.

To demonstrate the validity of the phenotypic detection approach basedon volume regulation, we performed a prolonged application of glutamateat a concentration of 50 μM during 90 seconds. The mean phase valuemeasured on four cell bodies is shown in FIG. 4(a), where the reagenthas been applied several times on the culture with an interval ofapproximately one hour. The phase signals presented correspond to thecells shown in FIG. 4(b). One can identify that among the four curvesshown, three present a strong phase signal drop of approximately 40°,while cell 4 retrieves the same mean phase value after a short period oftime of approximately 10 minutes, while having phase variations below10°. The strong phase drops can be interpreted as an irreversiblederegulation of the cell volume, which ultimately leads to cell death,while the recovery of the other signal corresponds to a rapid regulationof cell volume.

Brightfield color images of the culture, enabling the assessment throughthe trypan blue dye as a control experiment, are presented in FIGS.4(b-c), respectively at t=30 min before the excitotoxic stimulation andt=290 min, where non viable cells are identified by the blue staining.The staining confirms the interpretation derived from the phase signal,as the nuclei of cells 1 to 3 were stained at respectively t=116 min,t=207 min and t=252 min, confirming cell death, while cell 4 remainedunstained with a healthy morphology, which shows that this cell is stillviable several hours after the drug exposure.

Those results could be reproduced on several cultures, as shown in FIG.5., where slightly different parameters of stimulations were employed.In FIG. 5(a), a glutamate pulse of 90 seconds (25 μM) producedreversible responses in phase, comparable to the one of cell 4 in theprevious experiment. The viability of cells could be confirmed with thereagent up to 4 hours and 30 minutes after stimulation, time at whichthe experiment was interrupted, with all cells being unstained andhaving a healthy morphology similar to the one at the beginning of theexperiment. In FIG. 5(b), a pulse of 120 seconds (50 μM) producedirreversible phase drops of typically 40° to 50°. In a similar way, celldeath could be confirmed with the reagent approximately 4 hours afterthe stimulation (t=323 min for cells 1 and 2, and t=385 min for cell 3).Curves of both experiments presented in FIG. 5 are representative ofmeasurements on n=10 cells each.

One can identify from the different experiments presented above thatsignal dynamics for the different concentrations employed forstimulation are fairly comparable and occurring in time frames in orderof tens of minutes, time after which a steady-state is reached, eitherwith a strong phase drop, or with a phase value recovery, depending onthe viability of the cell considered. Furthermore, it is possible toidentify a reproducible behavior regards to excitotoxic stimulationconcentrations and durations, as a 50 μM, 90 s pulse generated mixedresponses between recovery and death in cultures, a 25 μM, 90 s pulseinduced only recovery curves, and 50 μM, 120 s induced irreversiblesignals. The responses to similar concentrations and durations could bereproduced on several cultures.

The different experiments presented above show a very good agreementbetween cell viability assessment through dye reagent and volumephenotype monitoring through DHM for cell death detection, although bothtechniques rely on very different detection methods. On one side, thetrypan blue reagent relies on the change of membrane permeability whichhappens during necrosis, occurring either as the primary cause of celldeath mechanism, or as secondary necrosis. The altered permeability ofthe membrane makes it possible for the dye to penetrate the cell, andtherefore stain the nucleus. This implies that both cell and nucleusmembranes are altered, classically creating a residual stain in the cellbody before staining clearly the nucleus. Many different dyes assessingcell viability rely on the cell membrane permeability as the primaryindicator, such as trypan blue or propidium iodide (PI). This type ofstaining is commonly considered as aspecific, as they are not capable ofdistinguishing between primary necrosis and apoptosis, during whichsecondary necrosis occurs at a late stage in cell cultures.

On the other hand, the proposed method example in this feasibilitydemonstration relies on the dynamic measurement of cell morphologyphenotypes, and more specifically cell volume regulation. DHM providesthe possibility of measuring dynamically this phenotypic parameter onthe whole field of view in real time, making it possible to monitor thecell morphology with a time resolution in milliseconds.

CONCLUSION

Cell volume regulation measurement, and more generallyoptically-retrieved phenotypes linked to homeostasis provide a simplemean for early and time-effective cell death detection, while cellviability assessment through staining reagents may take up to severalhours after the drug application. Employing different phenotypesprovided by the DHM measurement or other optical measurement meansenables a faster detection. First, one can identify that if cells didnot yet regulate regarding its environment during a given time after thedrug application, it will not be possible for regulation mechanisms tooccur at a sufficient speed to recover from the shock. Furthermore, asexposed for example in the previous feasibility demonstration with thevolume phenotype, one can also see that the magnitudes of the phenotypechanges are dramatically different for cells regulating, e.g. theirvolumes, and cells for which death mechanisms were triggered. In thelatter case, larger phase changes are occurring, while in the case ofcells recovering, phase decrease is finally compensated by theregulation mechanisms, so that small changes only are observed. Thosecriteria enable an easy diagnostic for cell viability through dynamicmeasurements of phenotypes, making it possible to define thresholds atwhich the cell will not recover, and for which cell death mechanismsstarted.

REFERENCES

-   Duprez, L. et al. “Major cell death pathways at a glance”, Microbes    Infect. 11(13), 1050-1062 (2009).-   Bortner, C. and Cidlowski, J., “The role of apoptotic volume    decrease and ionic homeostasis in the activation and repression of    apoptosis,” Pflug. Arch. Eur. J. Physiol. 448(3), 313-318 (2004).-   Colomb, T., E. Cuche, N. Aspert, J. Kühn, P. Marquet, C.    Depeursinge, F. Montfort, F. Charrière, A. Marian, S. Bourquin et    al. Wave Front Sensing Method And Apparatus, WO2006090320 (2006).-   Cuche, E., and C. Depeursinge. Method for simultaneous amplitude and    quantitative phase contrast imaging by adjusting reconstruction    parameters for definition of digital replica of reference wave and    aberration parameters correction digitally, EP1119798 (2000).-   P. Marquet, E. Cuche, C. Depeursinge, P. Magistretti, Apparatus and    Method for Digital holographic imaging, EP1451646 (2003),

1-7. (canceled)
 8. An apparatus for monitoring cell viability in asample, said apparatus comprising: an emitter for providing anelectromagnetic wave for interaction with at least one cell of saidsample; a measurement device configured to measure at least one opticalproperty of said cell, said at least one optical property being probedwith said electromagnetic wave and said at least one optical propertybeing the phase of said electromagnetic wave interacting with said atleast one cell; an applicator device for carrying out a cell environmentperturbation of said at least one cell; a digital holographic microscopeto make temporally resolved measurements of said optical property tomonitor a cell regulation behaviour of said at least one cell, said cellregulation behaviour being a cell mechanism activated to restore ahomeostatic equilibrium of said at least one cell after said cellenvironment perturbation; and a processer configured to determine thatsaid at least one cell is viable when a change in the measured opticalproperty, due to said cell environment perturbation, is reversible suchthat the measured optical property recovers to a level comparable tothat before said cell environment perturbation, which indicates arestoration of cell homeostatic equilibrium by cell regulation.
 9. Theapparatus according to claim 8, wherein the apparatus is configured tomeasure from said phase at least one selected from the group consistingof: optical path length of cells, index of refraction of cells,thickness of cells, volume of cells, surface of cells, morphology ofcells, protein content of cells, and water concentration of cells. 10.The apparatus according to claim 8, wherein the processor is furtherconfigured to determine cell death has occurred when a change in themeasured optical property, due to said cell environment perturbation, isnon-reversible such that the measured optical property does not recoverto a level comparable to that before said cell environment perturbation.11. The apparatus according to claim 10, wherein the processor isconfigured to evaluate from said phase at least one selected from thegroup consisting of: optical path length of cells, index of refractionof cells, thickness of cells, volume of cells, surface of cells,morphology of cells, protein content of cells, and water concentrationof cells.
 12. The apparatus according to claim 8, wherein said processoris configured to process the temporally resolved measurements of saidoptical property to analyze at least one phenotype selected from thegroup consisting of apoptosis, necrosis, and autophagy.
 13. An apparatusfor monitoring cell viability in a sample, said apparatus comprising: anemitter for providing an electromagnetic wave for interaction with atleast one cell of said sample; a measurement device configured tomeasure at least one optical property of said cell, said at least oneoptical property being probed with said electromagnetic wave and said atleast one optical property being the phase of said electromagnetic waveinteracting with said at least one cell; an applicator device forcarrying out a cell environment perturbation of said at least one cell;at least one device selected from the group consisting of aninterferometer, a wavefront sensor, and a phase-contrast microscope tomake temporally resolved measurements of said optical property tomonitor a cell regulation behaviour of said at least one cell, said cellregulation behaviour being a cell mechanism activated to restore ahomeostatic equilibrium of said at least one cell after said cellenvironment perturbation; and a processor configured to determine thatsaid at least one cell is viable when a change in the measured opticalproperty, due to said cell environment perturbation, is reversible suchthat the measured optical property recovers to a level comparable tothat before said cell environment perturbation, which indicates arestoration of cell homeostatic equilibrium by cell regulation.
 14. Theapparatus according to claim 13, wherein the apparatus is configured tomeasure from said phase at least one selected from the group consistingof: optical path length of cells, index of refraction of cells,thickness of cells, volume of cells, surface of cells, morphology ofcells, protein content of cells, and water concentration of cells. 15.The apparatus according to claim 13, wherein the processor is furtherconfigured to determine cell death has occurred when a change in themeasured optical property, due to said cell environment perturbation, isnon-reversible such that the measured optical property does not recoverto a level comparable to that before said cell environment perturbation.16. The apparatus according to claim 15, wherein the processor isconfigured to evaluate from said phase at least one selected from thegroup consisting of: optical path length of cells, index of refractionof cells, thickness of cells, volume of cells, surface of cells,morphology of cells, protein content of cells, and water concentrationof cells.
 17. The apparatus according to claim 13, wherein the processoris configured to process said temporally resolved measurements of saidoptical property are carried out to analyze at least one phenotypeselected from the group consisting of apoptosis, necrosis, andautophagy.